Aminoacy-tRNA synthetases establish the genetic code through aminoacylation reactions that link specific amino acids to tRNAs that bear triplet anticodon sequences. The universal distribution of these enzymes across the phylogenetic tree suggests that they are among the oldest proteins to have developed specificity towards amino acids and tRNAs. The specificity of cysteinyl-tRNA synthetase (CysRS) is extraordinary- even the simple replacement of the thiol of the substrate cysteine with the hydroxyl of serine, or a single substitution in tRNA cys can cause reduction in activity of a million-fold or more. Although recent studies have provided major insights into the substrate specificity of CysRS, this is not sufficient to understand specificity in broader evolutionary terms. For example, recent studies have identified a CysRS embedded within the sequence framework of a prolyltRNA synthetase (ProRS) that has the ability to activate both proline and cysteine and catalyze aminoacylation of tRNA with proline and with cysteine. The dual-specificity ProRS has challenged the view of one synthetase for one amino acid and raised many fundamental questions about synthetase specificity. In addition, recognition of tRNAcys through indirect readout of structural features has been established for bacterial, but not eucaryotic, CysRS. Such an indirect readout can have major impact on our understanding of specificity well beyond that obtained from analysis of direct contacts in tRNA-synthetase interactions. The difference between the bacterial and eucaryotic recognition also provides the basis for developing species-specific inhibitors of aminoacylation. Further, emerging crystal structures of E. coli CysRS have now offered a novel opportunity to address the outstanding question of how this enzyme recognizes cysteine and discriminates against the closely similar serine without an editing mechanism. This investigation is timely and promises new insight into molecular medicine that targets disorders in amino acid metabolism. Three specific aims are proposed: (1) to study recognition of tRNAcys by the dual-specificity ProRS of the halophilic archaeon Halobacteriurn halobium, (2) to study the thermodynamic and structural contribution of indirect readout of tRNA structural motifs in aminoacylation, and (3) to study the molecular basis of the exquisite specificity of E. coli CysRS for its ability to distinguish cysteine from serine. These studies shall shed new light on the molecular interactions responsible for the accurate translation of the genetic code.